The toxicity (or side effects) that patients suffer as a result of anti-cancer drugs taken during the process of chemotherapy remains the biggest enemy associated with cancer treatment. The therapeutic index (TI) of an anti-cancer agent is defined as a ratio of the most tolerated dose to the effective dose. It is known that most anti-cancer agents clinically used today have a very narrow TI range (1 to 1.2). In other words, the toxic effects of most anti-cancer agents in patients receiving chemotherapy are unavoidable at the effective dose levels because of the narrow therapeutic index associated with the anti-cancer agents. The differences in toxicity between agents are the grades of severity and types of suffering. Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents for the systemic and palliative treatment of patients with cancers arising from the gastrointestinal tract, breast, head, and neck. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU. A DPD deficiency is increasingly recognized as an important pharmacogenetic disorder in the etiology of severe 5-FU associated toxicity. It has been reported that cancer patients who were genetically heterozygous or homozygous for a mutant allele of the gene encoding DPD suffered from severe toxicity, including death, following the administration of 5-FU. Van Kuilenburg A B P, Haasjes J, Richel D J et al. Clinical implications of dihydropyrimidinedehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clinical Cancer Res 2000, 6: 4705-4712. Van Kuilenburg A B P, Muller E W, Haasjes J et al. Lethal outcome of a complete dihydroprimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14+1G>A mutation causing DPD deficiency. Clin Cancer Res 2001, 7: 1149-1153. Platinum agents are also commonly used for treatment. A substantial body of literature documents the side effects of platinum compounds. Cisplatin has multiple toxicities: nephrotoxicity, neurotoxicity, ototoxicity, nausea and vomiting. DeVita V T, Hellman S, and Rosenberg S A. Cancer, Principles and Practice of Oncology, 7th Ed, Lippincott Williams & Wilkins 2005, 335. The nephrotoxicity of cisplatin almost led to its abandonment, until the introduction of aggressive hydration by Cvitkovic and his coworkers, which prevented the development of acute renal failure. Cvitkovic E, Spaulding J, Bethune V, et al. Improvement of cis-dichlorodiamineplatinum (NSC 119875): therapeutic index in an animal model. Cancer 1977, 39, 1357. Hayes D, Cvitkovic E, Golbey R, et al. High dose cis-platinum diamine dichloride: amelioration of renal toxicity by mamnitol diuresis. Cancer 1977, 39, 1372. The toxicity of cisplatin was considered by some to be a driving force in history both in the search for less toxic analogues and for more effective treatment for its side effects. Myelosuppression, which is not usually severe with cisplatin, is the dose-limiting toxicity of carboplatin. Evens B, Raju K. Calvert A, et al. Phase II study of JM8, a new platinum analogue, in advanced ovarian carcinoma. Cancer Treat. Rep., 67, 997, 1983. The dose-limiting toxicity of oxaliplatin is sensory neuropathy—a characteristic of all DACH-containing platinum derivatives. DeVita V T, Hellman S, and Rosenberg S A. Cancer, Principles and Practice of Oncology, 7th Ed, Lippincott Williams & Wilkins 2005, 335. Alkylating agents also play important roles in cancer treatments. Each alkylating agent is associated with a specific toxicity and is not discussed individually. The following toxicities are common to the alkylating agents as a class: hematopoietic toxicity, gastrointestinal toxicity, gonadal toxicity, pulmonary toxicity, alopecia, teratogenicity, carcinogenesis, and immunosuppression. DeVita V T, Hellman S, and Rosenberg S A. Cancer, Principles and Practice of Oncology, 7th Ed, Lippincott Williams & Wilkins 2005, 335. The usual dose-limiting toxicity for an alkylating agent is hematopoietic toxicity. Topoisomerase-interactive agents have increasingly gained attentions from clinical oncologists for their unique action mechanism. Toptecan is a semi-synthetic product from natural-occurring 10-hydroxycamptothecin and is indicated in the second-line treatment of advanced refractory ovarian, and small cell lung cancers, and it also has been active in the treatment of hematologic malignancies, including myelodysplastic syndromes and multiple myeloma. Huinink W, Gore M, Carmichael J, et al. Topotecan versus paclitaxol for the treatment of recurrent epithelial ovarian cancer. J Clin Oncol 1997, 15, 2183; Schiller J H, Adak S, Cella D, et al. Topotecan versus observation after cisplatin plus etoposide in extensive-stage small-cell lung cancer: E7593-a phase II trial od the Eastern Cooperative Oncology Group. J Clin Oncol 2001, 19, 2114; von Pawel J, Schiller J H, Shepherd F A, et al. Topotecan versus cyclophosphamide, doxorubicin, and vincristine for the treatment of recurrent small-cell lung cancer. J Clin Oncol 1999, 17, 658, Pizzolato J F, Saltz L B. The camptothecins. Lancet 2003, 361, 2235. The dose-limiting toxicity of this agent is myelosuppression. Although topotecan has been combined with a variety of other treatments, including radiation, cisplatin, paclitaxol, and doxorubicin in clinical trials, none of these combinations has achieved any routine use in clinical oncology. This may be due, in part, to the frequent myelosuppresive toxicity of topotecan that has made it difficult to combine in high doses with other bone marrow-suppressive agents. Miller A A, Lilenbaum R C, Lynch T J, et al. Treatment-related fatal sepsis from topotecan/cisplatin and topotecan/paclitaxol. J Clin Oncol 1996, 14, 1964. Irinotecan is also a semi-synthetic product from camptothecin family. This compound is indicated as a single agent or in combination with florouracil and leucovorin in treating patients with colorectal cancers and also found active in small cell lung cancer when given in combination with cisplatin. This combination has been found active in non-small cell lung cancer as well. Saltz L B, Cox J V, Blanke C, et al. Irinotecan plus florouracil and leucovorin for metastatic colorectal cancer. Irinotecan study group. N Engl J Med 2002, 343, 905; Douillard J Y, Cunningham D, Roth A D, et al. Irinotecan combined with florouracil compared with florouracil alone as first-line treatment for metastatic colorectal cancer: a multicenter randomized trial. Lancet 2000, 355, 1041; Pizzolato J F, Saltz L B. The camptothecins. Lancet 2003, 361, 2235. The dose-limiting toxicities of irinotecan are neutropenia and delayed-onset diarrhea, and its uses in clinical oncology are thus limited too. All other miscellaneous anti-cancer agents, including recently marketed erbitux and avastin, may be used for a limited number of specific treatments, but are also associated with toxicities. Thus, it is still a great challenge for cancer researchers and clinical oncologists to find better agents with a wider therapeutic index for treatment.
Camptothecin, a cytotoxic alkaloid first isolated from the wood and bark of Camptotheca Acuminata (Nyssaceae) by Wall and his coworkers (J. Am. Chem. Soc. 88, 3888, 1966), was shown to have antitumor activity against the mouse leukemia L 1210 system. The structure of camptothecin, an alkaloid which has a commonly occurring indole alkaloid group (Heckendorf et al., J. Org. Chem. 41, 2045, 1976), is shown below as Formula (X).

This compound (“CPT”) has a pentacyclic ring system with only one asymmetrical center in ring E with a 20(S)-configuration. The pentacyclic ring system includes a pyrrolo[3, 4-b] quinoline moiety (rings A, B and C), a conjugated pyridone (ring D), and a six-membered lactone (ring E) with an α-hydroxyl group. Camptothecin was of great interest from the time of its initial isolation due to its noteworthy activity in the mouse leukemia L 1210 system. Earlier data for the antitumor activity of camptothecin were obtained by employing experimentally transplanted malignancies such as leukemia L 1210 in mice, or Walker 256 tumor in rats (Chem. Rev. 23, 385, 1973, Cancer Treat. Rep. 60, 1007, 1967). Subsequent clinical studies showed that this compound was not usable as an anticancer agent in vivo due to its high toxicity. Camptothecin itself is insoluble in water. Therefore, camptothecin was evaluated clinically as a water-soluble sodium carboxylate salt in the early times. This form of camptothecin produced severe toxicity and seemed devoid of anticancer activity (Gottlieb et al., Cancer Chemother. Rep. 54, 461, 1970, and 56, 103, 1972, Muggia et al., Cancer Chemother. Rep. 56, 515, 1972, Moertel et al., Cancer Chemother. Rep. 56, 95, 1972, and Schaeppi et al., Cancer Chemother. Rep. 5:25, 1974). These results caused the discontinuation of phase I trials. Continued evaluation of this agent showed that the sodium carboxylate salt is only 10% as potent as the native camptothecin with the closed lactone ring intact (Wall et al., In International Symposium on Biochemistry And Physiology of The Alkaloids, Mothes et al., eds., Academie—Verlag, Berlin, 77, 1969, Giovanella et al., Cancer res. 51, 3052, 1991). In addition, important parameters for antitumor activity in the camptothecin family have been established (Wall et al., Ann. Rev., Pharmacol Toxicol 17, 117, 1977). These results indicate that an intact lactone ring E and α-hydroxyl group are essential for antitumor activity.
In the middle 1980s it was found that the molecular target of camptothecins was the novel nuclear enzyme topoisomerase I. Hsiang Y H, Liu L F. Identification of mammalian DNA topoisomerase I as an intracellular target of anticancer drug camptothecin. Cancer Res 1988, 48, 1722. At approximately the same time, several new water-soluble camptothecin derivatives, including two compounds (topotecan and irinotecan) discussed earlier, were prepared and biologically evaluated. The subsequent clinical evaluations of the two compounds demonstrated the predictable toxicities and meaningful anticancer activity. Takimoto C H, Arbuck S G. Topoisomerase I targeting agents: the camptothecins. In: Chabner B A, Longo D L, eds., Cancer therapy & biotherapy: principles and practice, 3rd ed. Philadelphia: Lippincott Williams & Wilkins 2001, 579. Topotecan was approved in 1996 as second-line treatment for advanced ovarian cancer, and it later gained the indication for treating patients with refractory small cell lung cancer. At exactly the same time, irinotecan was registered for treating 5-florouracil-refractory advanced colorectal cancer. This actually represented the first new agent to gain approval for treating this disease in the United States in nearly 40 years.
In 1989, Giovanella et al. found that some of the non-water soluble derivatives of camptothecin have high antitumor activity against xenografts of human tumors (Giovanella et al., Science, 246, 1046, 1989). It was also shown that administration of camptothecin with closed lactone ring is superior to injections of water-soluble carboxylate salt (Giovanella et al., Cancer Res., 51, 3052, 1991). These findings further confirmed the importance of the intact lactone ring to biological activity.
Ring opening of 20(S)-camptothecin leads to much more potent anticancer activity in mice than in humans. In effect, CPT administered intramuscularly (“i.m.”) subcutaneously (“s.c.”), and intrastomach (“i.s.”) has proven to be a very potent anticancer agent against human tumors in mice, i.e., when growing as xenotransplants in nude mice (Giovanella et al., Cancer Res. 51:3052, 1991). However, when tumors were treated with CPT in humans, a lower degree of anticancer activity in humans, than in mice, was exhibited (Stehlin et al., In Camptothecins: New Anticancer Agents, 1995, CRC Press, pp. 59-65).
The same phenomenon was observed with other CPT-derivatives. In mice, 9-nitrocamptothecin (“9NC”) has proven to be 2-3 times more potent than CPT against human tumor xenografts causing the total eradication of all the human malignancies treated (Pantazis et al., Cancer Res. 53:1577, 1993; Pantazis et al., Int. J. Cancer 53:863, 1995).
Ring opening is particularly problematic in that camptothecins exist in two distinct forms. The naturally-occurring camptothecin has an S-configuration and is 10 to 100 times more biologically active than the R-isomer. The S-configured lactone form is thought to be required for anti-tumor activity, and the carboxylate form usually relates to clinical toxicities. The molecule exists in equilibrium in aqueous solution. This equilibrium is pH-dependent. At physiological pH, i.e., 7 or above, the equilibrium equation is as follows:

The hydrolysis reaction of the biological active lactone ring of camptothecins with water at higher pH gives the biologically inactive open form. Additionally, the hydrolysis problem with CPT and its analogs is exacerbated in human blood because the predominant human serum albumin (HSA) preferentially binds to the carboxylate form, which shifts the lactone/carboxylate equilibrium toward the inactive form (J. Biochem., 212, 285-287, 1993; Biochemistry, 33, 10325-10336, 1994; Biochemistry, 33, 12540-12545, 1994). Accordingly, preserving the lactone ring of the molecule for a sufficient time for the tumor cells to cycle through the S-phase is a major challenge and has been the focus of a considerable amount of research.
A number of attempts have been made to provide derivatives of camptothecin having greater biological activity and enhanced stability. Many of these compounds are the products of modifications on the A, B, and C rings of the molecule, but few of these modifications have enhanced the stability of the lactone ring under physiological conditions. Other approaches have been more successful. For instance, acylating of 20-OH group provides a useful tool for the protection of lactone ring E. Wall et al., U.S. Pat. No. 4,943,579, describes several acylated camptothecin compounds having water solubility, although the lactone may not remain intact under physiological conditions. U.S. Pat. No. 5,968,943 to Cao et al. discloses CPT-derivatives which are effective antitumor agents.
A number of different reactions are reported in literature for preparing camptothecin esters.
Direct acylation of camptothecin with organic acid anhydrides with pyridine as catalyst was employed for preparing alkyl and alkenyl camptothecin esters (as shown above). This reaction usually gives high yields, but the availability of organic acid anhydrides restricts the scope of the reaction.
A dicyclohexylcarbodiimide (DCC)/dimethylaminopyridine (DMAP) reagent system was therefore, used for acylation reactions of carboxylic acids with alcohols and thiols. Previously, a method was used to prepare aromatic camptothecin esters (as shown below).
This procedure, however, gives good reaction yields only when the carboxylic acids are very electrophilic. When the acids are less electrophilic, the reaction gives low yield or no expected product at all. For example, when using propionic acid to prepare camptothecin propionate with this procedure, the ester product was essentially not obtained and the starting camptothecin was almost 100% recovered.
Nonanoic chloride was also used as an acylating agent to esterify camptothecin with pyridine as an HCl-trapping agent in methylene chloride. The reaction (as shown below) occurred with low yield (6%).

As the inherent structural features of camptothecin provide an ideal platform for cancer researchers to modify the structure for obtaining better anti-cancer agents, many different camptothecin esters have been synthesized and evaluated. It has been found that the esterification products largely increased the biological life of the molecules in the body. It has also been shown that treatment of human tumors grown in xenografts in nude mice with synthetic camptothecin esters is effective, and toxicity in mice is minimal. Cao, Z.; Pantazis, P.; Mendoza, J.; Early, J.; Kozielski, A.; Harris, N.; Vardeman, D.; Liehr, J.; Stehlin, J.; Giovanella, B. Ann. N.Y. Acad. Sci. 2000, 922, 122; Cao, Z.; Pantazis, P.; Mendoza, J.; Early, J.; Kozielski, A.; Harris, N.; Giovanella, B. Acta Pharmacologica Sinica 2003, 24, 109.
While many methods for preparing camptothecin esters exist, however, each procedure has certain restrictions as discussed above. Therefore, there is still a need to develop camptothecin esters which retains the anti-tumor activity of the mother compound, CPT, has much lower toxicity than CPT, and which can be produced by alternative procedure(s) for preparing camptothecin esters.